Detailed studies of postnatal neurons of the mammalian visual system would be aided by examining the chemically-gated ion channels of identified cells in a controlled in vitro environment. For this purpose cultures of rodent retinal ganglion cells, identified with specific fluorescent probes, are used in this laboratory. The specific aims of this proposal are -- (i) To study physiologically and pharmacologically the effects of putative neurotransmitters and modulators on solitary mammalian retinal ganglion cells in culture. The effect of various substances on both whole-cell and single-channel currents will be monitored using the patch-clamp technique. If calcium current or calcium-activated currents appears to be affected by an agent, then calcium will be quantitated using imaging techniques with the calcium-sensitive dye, fura-2. Each of the substances to be studied is a likely candidate to affect ganglion cells because of its presence in amacrine and bipolar cells which normally synapse on the ganglion cells in the intact retina. These substances include acetylcholine, GABA, glycine, glutamate, dopamine, and indoleamines, as well as many peptides such as leu-enkephalin, substance P, somatostatin, cholecystokinin, neurotensin, and vasoactive intestinal peptide (VIP). (ii) To identify the transmitters used at chemical synapses onto retinal ganglion cells. The substances found to affect solitary ganglion cells will be tested for their effects on endogenous synaptic activity which occurs in ganglion cells located among clusters of other retinal cells in the cultures. In this manner an attempt will be made to discern the identity of the physiologically-relevant transmitters and modulators. (iii) To quantify trophic effects of the putative neurotransmitters and modulators and their selective blocking agents by monitoring survival and regeneration of processes by retinal ganglion cells in culture. Preliminary experiments have already suggested that nicotinic cholinergic antagonists and opiate analogs influence the regeneration of neurites by retinal ganglion cells. Ionic mechanisms underlying process regeneration may be uncovered in these experiments since the ionic basis for the current induced by each substance will have been studied in the patch clamp experiments. The long-term goals of this project are to further the understanding of synaptic communication between central neurons in the mammalian visual system and to increase neuronal survival and the potential for process regeneration.